In utero bone marrow transplantation is currently being investigated as a therapeutic modality to correct inherited lymphohematopoietic disorders. While engraftment has been observed in both animal and human fetal recipients of in utero transplanted hematopoietic cells the level of engraftment in non-anemic recipients has generally been low. Our preliminary data in W locus defective mice injected in utero with adult bone marrow cells demonstrates that up to 10-15% of cells with long-term (currently > 2.5 months post-injection) and multi-lineage (erythroid; lymphoid; myeloid) properties are of donor origin. Using a mouse model of in utero transplantation, we will ask: 1. is in utero engraftment limited by the numbers of adult PHSCs infused as compared to resident fetal PHSCs ?; 2. are fetal PHSCs qualitatively superior to adult PHSCs in providing the fetus with hematopoietic cell populations with post- natal repopulating potential ? If so, are these differences due to homing patterns or responsiveness to the particular milieu of cytokines produced by the fetus? We will address these 2 issues by injecting anemic (W locus mutant) mice with 2 types of donor grafts, each distinguishable from each other and from the fetal recipient by FACS analysis and isoenzyme typing. A series of donor hematopoietic cell enrichment techniques will be assessed that will concentrate to varying degrees PHSC and/or committed progenitor cells from one type of donor graft that can be competed in vivo with the second type of donor graft and the fetal recipient. In utero migration of adult versus fetal PHSC will be investigated using in situ immunohistochemistry and FACS, while early post-injection proliferation in situ will be assessed by using lipophilic dyes that irreversibly bind to lipid bilayers in the cell membrane by immunohistochemistry and by FACS analysis of engrafted cell populations. We will attempt to increase in utero engraftment and post-natal reconstitution by preincubating the enriched donor BM fractions with cytokines in a stroma non-contact or a stroma-free setting to diminish the effect of negative regulators on progenitor cell proliferation. In addition, we will coadminister non-alloreactive (F1) T-cells as a cellular source of cytokines in utero. Once we have obtained sufficient engraftment in anemic mice, we will progress to studies in non-anemic mice. These studies together will form the basis of important methods to obtain in utero engraftment of hematopoietic progenitors in larger animals and humans.